EFFICACY OF CENTELLA ASIATICA EXTRACT IN THE MANAGEMENT … · EFFICACY OF CENTELLA ASIATICA...

www.wjpps.com Vol 5, Issue 12, 2016.

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Vuppala et al. World Journal of Pharmacy and Pharmaceutical Sciences

EFFICACY OF CENTELLA ASIATICA EXTRACT IN THE

MANAGEMENT OF CRACKED FEET: IN VITRO AND CLINICAL

EVIDENCE

Muhammed Majeeda,b

, Priti Vaidyanathana, Lakshmi Mundkur

a, Shaheen Majeed

a,b,

Pratiksha Sablea and Kian Kumar Vuppala

c*

aSami Labs Limited, Peenya Industrial Area, Bangalore – 560 058, Karnataka, India

bSabinsa Corporation, 750 Innovation Circle, Payson, UT 84651, USA

cClinWorld Private Limited, # 19/1&19/2, I Main, II Phase, Peenya Industrial Area,

Bangalore – 560 058, Karnataka, India.

ABSTRACT

OBJECTIVE: The aim of the study was to evaluate the efficacy and

safety of a formulation, Centellin® CG foot care cream (CCFC)

containing 2% Centella asiatica leaf extract, in the management of

cracked heels through cell culture and human clinical studies.

METHODS: Human dermal fibroblast (adult) primary cell line and

mouse macrophage cell line J774 were used to evaluate cell

proliferation, collagen enhancement and wound healing ability of

CCFC. For the clinical study, 24 female subjects suffering from

cracked heels were enrolled to topically receive CCFC, twice daily for

4 weeks (28 days). Subjects underwent evaluation of efficacy

parameters such as assessment of cracked heels, effect of skin

moisturizing and photographs before and at the end of the treatment.

Subjects revisited the study site on day 7, 14, 21 and 28 for testing of efficacy parameters.

RESULTS: CCFC has a beneficial effect on cell proliferation, collagen synthesis and

significantly improves wound healing in the fibroblast scratch wound model. A significant

reduction in number of cracks was seen at the end of the study. There was significant increase

in skin moisturizing and visually acceptable changes in the treatment photographs were

recorded. Acceptability and compliance to the use of the formulation was good. No adverse

events were reported during the study. CONCLUSION: The findings demonstrate that

CCFC helps in healing of cracks, reducing scaling and pain. None of the volunteers

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.041

Volume 5, Issue 12, 983-994. Research Article ISSN 2278 – 4357

Article Received on

05 October. 2016,

Revised on 25 October 2016,

Accepted on 15 Nov. 2016

DOI: 10.20959/wjpps201612-8213

*Corresponding Author

Kian Kumar Vuppala

ClinWorld Private

Limited, # 19/1&19/2, I

Main, II Phase, Peenya

Industrial Area, Bangalore

– 560 058, Karnataka,

India.


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experienced any hypersensitivity reactions. Cell culture data demonstrates that CCFC

facilitates wound healing process by stimulating cell proliferation and collagen synthesis.

Therefore, it may be concluded that the CCFC is safe and efficacious in management of foot

cracks.

KEY WORDS: Cell culture, Centella asiatica, cracked heels, skin barrier, skin physiology,

wound healing.

INTRODUCTION

Cracked heels, also known as heel fissures, are a common foot problem characterized by

yellowing of skin on the heel of the foot; callused, hard skin growth, flaky patches, cracked

and peeling skin. Leading causes include standing for prolonged periods of time (specifically

on hard floors), use of open-heeled shoes which cause the heel to expand and increase

pressure and medical conditions like diabetes which facilitate dry skin formation. Skin

conditions such as psoriasis and eczema may also cause cracked heels and feet. Obesity can

also increase the chances of cracks by putting more pressure on fat pads under the heel

thereby causing it to expand. Feet that are constantly in close contact with water tend to lose

their natural oil and turn dry and rough. In extreme cases, cracked heels can get infected and

lead to cellulitis [1]

.

Herbal extracts have played a vital role in numerous cultures for the prevention and

management of various health conditions. Centella asiatica is a perennial herbaceous creeper

which belongs to the Umbelliferae (Apiceae) family and has extensively been mentioned in

Ayurveda. The active constituents in this herb include triterpenoid saponins, flavanoids,

volatile oils, amino acids, phytosterols, sugars and tannins [2]

. The extract used in the current

study is Centellin® CG, standardized to contain 40% triterpenoidal saponins which are made

up of constituents like asiaticosides, centellosides, madecassoside, thankuniside, isothankunic

acid, centellose, asiatic acid, centellic acid, madecassic acid, brahmoside, brahminoside and

brahmic acid.

Research articles published in recent times have substantiated the use of Centella asiatica in

various conditions including wound healing. In vivo studies report enhanced cell growth,

collagen synthesis around wound sites and quicker epithelialization. A specific component of

Centella asiatica, asiaticoside, is reported to increase the tensile strength of skin that newly

forms around wounds and even stimulate angiogenesis [3]

.


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Considering the extensive traditional knowledge and modern scientific validation around this

herb, in the current study it is proposed that Centellin® CG foot care cream (CCFC)

containing 2% Centella asiatica leaf extract provides significant relief to patients suffering

from cracked heels within 28 days of use. Additional in vitro data has been reported in this

study to validate the mechanisms involved.

MATERIAL METHODS

Formulation Details

CCFC has the following constituents (%w/w): Purified Water-83.30; Disodium EDTA-0.05;

Carbomer-0.20; Glyceryl stearate & PEG100 sterate-3.00; Cetyl alcohol-2.00; Isopropyl

Palmitate and Pentaerthirityl Tetraisostearate-2.00; Nonionic Emulsifying Wax-3.00;

Caprylic Capric triglycerides-2.00; Pentaerthirtyl Tetra-di-t-butyl hydroxyhydrocinnamate-

0.20; Centellin® CG-2.00; Amino Methyl Propanol-0.10; Cyclopentasiloxane, Dimethiconol,

Dimethicone Crosspolymer and Phenyltrimethicone Blend-0.75; Tocopheryl Acetate-0.20;

Phenoxy Ethanol & Ethyl hexyl Glycerin-0.80; Fragrance-0.40.

In vitro

Sample preparation

Stock solutions of CCFC were prepared in dimethyl sulfoxide (DMSO) at 2 mg/ml. Serial

dilutions of the stock was prepared to obtain the desired final concentrations.

Cell culture

Human dermal fibroblast (adult) primary cell line (HDFA) and mouse macrophage cell line

J774 were purchased from American Type Culture Collection (Rockville, MD). The cells

were cultured in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal

bovine serum (FBS) and 100 U/mL of penicillin and streptomycin in 5% CO2 at 37°C.

Cell viability/proliferation

Cell viability was determined by Sulforhodamine B (SRB) assay [4]

. Cells were seeded at a

density of 5 x 103 cells/well in DMEM with 10% FBS in 96 well plates and allowed to form

a monolayer in a humidified incubator at 37°C and 5% CO2 atmosphere for 24 hours. Cells

were treated with different concentrations of CCFC for 72 hours. Cell monolayers were fixed

with 10% (w/v) trichloroacetic acid and stained for 30 min with SRB, after which the excess

dye was removed by washing repeatedly with 1% (v/v) acetic acid. The protein-bound dye

was dissolved in 10 mM Tris base solution for OD determination at 510 nm using a


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microplate reader (TECAN Ltd, Männedorf, Switzerland). Cells in DMEM with 0.1% DMSO

were used as positive control while media without cells were used as negative control. The

results were expressed as percentages of control.

Collagen enhancement assay

HDFA cells were cultured as monolayer at 5 x 103 cells/well in DMEM with 2 % FBS in 96

well plate and treated with non toxic concentrations of CCFC for 72 hours. The culture

supernatant was collected to estimate collagen concentration. Cell layers were extensively

washed with phosphate-buffered saline (PBS) three times and then fixed with Bouin’s fluid

for 1 h at room temperature (RT). Collagen fibers were stained with 1% Picrosirius red in

saturated aqueous solution of picric acid at RT for 1 h. [5,6]

. The stained cell layers were

extensively rinsed three times with 0.01 N HCl to remove non-bound dye. Cells were lysed

with 0.1 N NaOH solutions and placed on a microplate shaker for 30min at RT. The optical

density at 540 nm was measured with a microplate (TECAN Ltd, Männedorf, Switzerland) to

quantify the collagen [7]

. The percentage collagen content (%) was calculated as 100 ×

(absorbance of treated sample / absorbance of control).

Wound healing assay

HDFA cells were seeded at a density of 5 x 104 cells/well in DMEM 2% FBS in 24 well plate

to form a monolayer of fibroblasts [8]

. When the cells were confluent, artificial wounds were

created in the monolayer by making a linear scratch in the centre of each well using the tip of

a sterile 200 µL plastic pipette tip. The cellular debris created from the scratch was removed

by gently washing the wells with PBS. The wounded cells were treated with the test

substances at different concentrations. Complete media and media supplemented with 0.5%

DMSO were used as control. Photomicrographs were taken after 24 hours using phase

contrast microscope to record wound closure. The culture supernatant was collected to

estimate cytokine concentration. The cells were then fixed in 4% formaldehyde and stained

with 1% crystal violet. The images were captured and area of wound closure was quantified

using Image-J software.

Enzyme-linked immunosorbent assay (ELISA)

The culture supernatant from the wound healing assay was used to estimate the levels of

transforming growth factor (TGF)-β and fibroblast growth factor as per instructions by the

manufacturer.


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Statistical analysis

Statistical analysis data are expressed as mean ± standard deviation. Differences in measured

variables between experimental and control group were assessed by two-tailed t-test. Results

were considered statistically significant at p< 0.05. Graph pad prism software was used for

statistical analysis (Graph-Pad, La Jolla, CA, USA).

Clinical study

Volunteer enrollment

Female volunteers were enrolled for this open-label study after signing informed consents.

Participants were informed about the cream composition and its role in treating cracked heels

prior to commencement of the study. Volunteers presenting cracks/fissures in feet, especially

in the heel region, associated with dryness, pain and roughness of the sole were included in

the study. Volunteers were included in the study if a “YES” was indicated to all the inclusion

criteria and “NO” to all of the exclusion criteria. Inclusion criteria: healthy female volunteers,

18 years of age or older with cracks in both feet. Exclusion criteria: a) History of known

dermatological diseases such as Psoriasis, eczema, ichthyosis vulgaris b) History of

peripheral vascular disease c) Use of topical steroids or moisturizers in the previous 2 weeks

d) Known hypersensitivity to topical preparations.

Study design

In this split controlled study, 12 participants received treatment on their right feet and 12 on

the left feet, with the untreated foot of each subject serving as a control. Volunteers suffering

from cracked heels visited on screening day. Subjects underwent physical examination and

assessment of heels. Screening visit was followed by baseline visit (Visit 1-Day 0) wherein

the subjects, based on the inclusion-exclusion criteria, were enrolled in the study. Subjects

were dispensed with study medication and were instructed to wash feet properly prior to

application of the cream twice daily. Subjects were informed to apply fingertip unit of CCFC

twice daily in the morning and night for a period of 28 days.

Subjects underwent assessment of efficacy parameters such as examination of cracked heels

and changes in moisturising the feet. Subjects returned to the study site on Visit 2 (Day 7),

Visit 3 (Day 14), Visit 4 (Day 21) and Visit 5 (Day 28). On Visit 2, Visit 3 & Visit 4 subjects

were dispensed with study medication. Efficacy assessments such as decrease in the number

of cracks, effect of skin moisturizing, visually acceptable changes in the treatment

photographs were conducted on Visit 5 (Day 28). All subjects were followed up at weekly


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intervals for a period of 4 weeks, and the symptom score evaluation was done during each

follow up visit. Pain assessment was conducted at each visit. After fourth week, subjects

independently rated the overall improvement on the following scale: 1-No changes, 2-

Average, 3-Good, 4-Excellent, 5-Extremly Excellent.

The primary efficacy endpoint was a decrease in the number of cracks, scaling, pain and

laxity of skin. The secondary safety endpoints were short and long-term safety, as assessed by

the incidence of adverse events.

RESULTS

Cell culture data

Viability and proliferation of human dermal fibroblasts

The viability of human fibroblast cells were assessed at different concentrations, of CCFC

ranging from 50 µg/ml to 0.19 µg/ml. At a concentration of 6 µg/ml the cream showed 41.8%

increase in proliferation (P=0.02) while at 3 µg/ml, the increase was 38.2% (P=0.03).

Significant increase in proliferative effect was observed up to 0.78 µg/ml (P<0.05). At higher

concentrations minor reduction in cell viability was observed (Fig.1).

Collagen synthesis in dermal fibroblasts

Wound healing is aided by the fibroblasts migrating towards the injured area which produce

collagen to increase tissue permeability. To understand the effect of CCFC on collagen

synthesis, treated dermal fibroblast cells were analyzed for their capacity to synthesize

collagen. The cream was found to significantly increase the collagen synthesis by 21%

(p=0.02) at 6 µg/ml, while it reduced to 3.48 % at 3 µg/ml and was negligible at 1.5 µg/ml

(Fig.2).

Wound healing in fibroblast scratch wound model

To ensure optimum activity of fibroblasts in wound closure, CCFC at 6 and 3 µg/ml was used

in the scratch wound healing assay. The ability of the cream treated human dermal fibroblasts

to cover the scratch wound compared to negative control is shown in Fig. 3. Significant

increase in wound closure (57.3% ± 7.9%, P=0.009 and 35.6± 6.9%, P=0.015) was observed

at 6 and 3 µg/ml respectively compared to untreated control. Fibroblast migration toward the

wound could be clearly observed in the treated wells. The concentration of TGF-β produced

in untreated control was below the detection limit. Treatment with 3 µg/mL of CCFC showed


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4 pg/mL of TGF-β, while the concentration increased to 79 pg/mL with 6 µg/mL of the

cream.

Clinical study

A total of 24 patients were screened and enrolled into the study. No participants were

excluded during screening. Out of 24 subjects between the age group of 18 to 40, 22 subjects

completed the study, two subjects dropped out citing personal reasons. Subject demographics

have been provided in Table 1.

There were no adverse events reported during the study and compliance to the use of the

formulation was good. Nature of the skin showed profound dryness at the screening visit

(Fig.4).

Significant reduction in the number of cracks and increase in the moisturization of the heel

was observed at the end of the study (Fig. 5).

Drastic visual change was noted in the photographs taken after 4-weeks treatment (Fig.6).

Overall product rating also showed good acceptance among the study subjects (Fig.7).

Table 1: Subject demographics

Demographics % of Respondents

Age

18-24 0

25-34 77.27

35-40 22.73


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DISCUSSION

Wound healing is a relatively complex process which can be divided into the following

stages: a) coagulation and haemostasis b) inflammation c) proliferation and d) wound

remodelling coupled with formation of scar tissue. Results from this study indicate that

CCFC has a beneficial effect on cell proliferation and collagen synthesis in dermal

fibroblasts. Collagen is one of the most abundant (25%) proteins in the body and is produced

by fibroblasts. In close contact with other proteins like elastin, collagen forms a matrix to

support components like sebaceous glands, blood vessels and dermal cells. Stimulation of

collagen synthesis during wound healing is vital to ensure cell multiplication and new tissue

formation[9]

. Few other studies have also underscored the potential of this herb and

demonstrated that in addition to improving cell proliferation, it enhanced DNA, protein and

collagen content of granulation tissues in rats. Greater cross linking of collagen was also

reported[10]

. Similar results were obtained when used in a gel formulation to treat open

wounds in rats[11]

. A report by Shetty et al. indicates that Centella asiatica can also counter

the inhibiting action that dexamethasone has on wound healing in rats[12]

.

A positive effect on wound healing obtained from cell culture studies was validated by

conducting an in-house clinical study. For most of the population, cracked heels are a

cosmetic problem, but when they deepen, the condition can turn painful. If left untreated, in

due course of time the pain can intensify and cause the cracks to bleed post which the skin is


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left highly susceptible to bacterial attack. Therefore, the Centellin®

CG foot care cream can

have moisturizing, analgesic and collagen boosting activity which helps in healing cracks and

reducing pain associated with it.

ACKNOWLEDGEMENTS

The Centellin® CG foot care cream was developed and supplied by Sami Labs Ltd,

Bangalore, India. The clinical trial was conducted by ClinWorld Pvt Ltd, Bangalore, India.

We express our sincere gratitude towards all the subjects who participated in the study.

Conflict of interest: None declared

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